Effects of retinoic acid on the concentrations of radioactive metabolites of retinol in tissues of rats maintained on a retinol-deficient diet

The effects of feeding retinoic acid for 2 and 6 days on the metabolism of labeled retinol in tissues of rats maintained on a vitamin A deficient diet was studied. The metabolites of retinol were analyzed by high performance liquid chromatography. Feeding retinoic acid for 2 days significantly reduced the blood retinol and retinyl ester levels without affecting the vitamin A content of the liver. In intestine and testis the content of labeled retinoic acid was decreased significantly by dietary retinoic acid. Addition of retinoic acid to the diet for 6 days resulted, in addition to decreased blood retinol and retinyl ester values, in an increase in the retinyl ester values in the liver. The accumulation of retinyl ester in the retinoic acid fed rat liver was accompanied by an absence of labeled retinoic acid. Kidney tissue was found to contain the highest levels of labeled retinoic acid, retinol, and retinyl esters; dietary retinoic acid did not alter the concentrations of these retinoids in the kidney during the experimental period. Since kidney retained more vitamin A when the liver vitamin A was low and also dietary retinoic acid did not affect the concentrations of radioactive retinoic acid in the kidney, it is suggested that the kidney may play a major role in the production of retinoic acid from retinol in the body.     

Pathways of retinol and retinoic acid metabolism in the rat

1. The metabolism of retinoic acid and retinyl acetate labelled with (14)C in various positions was studied after intravenous injection of physiological amounts of these compounds into retinol-deficient rats. 2. Analysis of the resultant radio-activity in the urine, carbon dioxide and faeces led to a postulation of the existence of three major pathways for the metabolism of these two compounds. 3. Evidence is presented that retinoic acid and retinol are metabolized by either the same or at least similar pathways and that retinol becomes oxidized to the carboxyl state before any degradation of the isoprenoid side chain occurs. 4. It is not possible to decide from these data whether retinoic acid is an intermediate in the retinol pathway. 5. Possible sites for the regulation of retinol metabolism are discussed.     

Characterization of retinoid metabolism in the developing chick limb bud

Retinoids (vitamin A derivatives) have been shown to have striking effects on developing and regenerating vertebrate limbs. In the developing chick limb, retinoic acid is a candidate morphogen that may coordinate the pattern of cellular differentiation along the anteroposterior limb axis. We describe a series of investigations of the metabolic pathway of retinoids in the chick limb bud system. To study retinoid metabolism in the bud, all-trans-[3H]retinol, all-trans-[3H]retinal and all-trans-[3H]retinoic acid were released into the posterior region of the limb anlage, the area that contains the zone of polarizing activity, a tissue possibly involved in limb pattern formation. We found that the locally applied [3H]retinol is primarily converted to [3H]retinal, [3H]retinoic acid and a yet unidentified metabolite. When [3H]retinal is locally applied, it is either oxidized to [3H]retinoic acid or reduced to [3H]retinol. In contrast, local delivery of retinoic acid to the bud yields neither retinal nor retinol nor the unknown metabolite. This flow of metabolites agrees with the biochemical pathway of retinoids that has previously been elucidated in a number of other animal systems. To find out whether metabolism takes place directly in the treated limb bud, we have compared the amount of [3H]retinoid present in each of the four limb anlagen following local treatment of the right wing bud. The data suggest that retinoid metabolism takes place mostly in the treated limb bud. This local metabolism could provide a simple mechanism to generate in a controlled fashion the biologically active all-trans-retinoic acid from its abundant biosynthetic precursor retinol. In addition, local metabolism supports the hypothesis that retinoids are local chemical mediators involved in pattern formation.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Retinoid metabolism during development of liver cirrhosis

The changes in retinoid metabolism have been documented in liver cirrhosis. However, the dynamic alterations in levels of this vitamin between circulation and liver during development of the liver cirrhosis are not well understood. The aim of this study was to measure retinoids in the liver and circulation in parallel, during and after development of cirrhosis induced by carbon tetrachloride and thioacetamide. Retinoid levels were measured by HPLC. A decrease in retinaldehyde and total retinol, together with an increase in retinoic acid was evident in liver from both carbon tetrachloride or thioacetamide treated rats within a month after initiation of treatment. Activity of enzymes involved in retinoid metabolism such as retinaldehyde oxidase, retinaldehyde dehydrogenase, and retinaldehyde reductase were decreased in the liver. In parallel, levels of retinol and retinaldehyde in the serum were increased while retinoic acid was decreased. This study indicates that during development of cirrhosis, there is reciprocal transfer of retinoid metabolites between the circulation and the liver.     

Retinoic acid formation from retinol in the human gastric mucosa: role of class IV alcohol dehydrogenase and its relevance to morphological changes

Alcohol dehydrogenase (ADH) participates in the formation of retinoic acid from retinol in various organs including the gastric mucosa. However, its clinical significance still remains to be clarified. In this study, we identified the ADH isoforms responsible for the retinoic acid formation among various ADH isoforms and examined associations among the ADH activities, the retinoic acid formation level, and morphological changes in the human gastric mucosa. Human gastric samples were endoscopically obtained from 67 male subjects. Morphological changes were assessed by the Sydney system and activities of class I, III, and IV ADH isoforms were determined in each specimen. In 26 cases, levels of all-trans retinoic acid (ATRA) formation from all-trans retinol were examined. Among activities of the three ADH isoforms, class IV ADH activity was solely associated with the ATRA formation level. This association was found even when subjects' age and Helicobacter pylori infection status were adjusted. As the degrees of inflammation, atrophy, and intestinal metaplasia increased, the class IV ADH activity as well as the potential for the ATRA formation decreased. Class IV ADH is a major enzyme in the retinoic acid supply in the human gastric mucosa, and the reduction of its activity was associated with decreasing retinoic acid supply and progression of inflammation, atrophy, and intestinal metaplasia in the gastric mucosa. In that retinoic acid is a key molecule for maintaining normal morphology, the reduction of class IV ADH activity may be involved in the pathogenesis of these morphological changes in the human gastric mucosa.     

Vitamin A and bone formation. Different responses to retinol and retinoic acid of chick bone cells in organ culture

The response of bone cells in organ culture to retinol and retinoic acid was studied. Both stimulated incorporation of [3H]thymidine into DNA by 16-day embryonic chick calvaria, but the time-course of the responses differed; the peak responses to retinol and retinoic acid occurred at about 18 h and 48 h, respectively. Although retinol inhibited chick bone collagen synthesis retinoic acid had no effect, but it did stimulate non-collagenous protein synthesis, whereas the effect on the latter of retinol was, if anything, inhibitory. When present with retinol, retinoic acid was able to attenuate the inhibitory effect of the former on chick bone collagen synthesis, but preincubation with retinoic acid had no such effect. In neonatal murine calvarial cultures, retinoic acid inhibited collagen synthesis selectively in the same manner as did retinol. The ability of chick osteoblasts to respond differently to retinol and retinoic acid suggests that both forms of the vitamin may have a role in bone formation and that their intracellular models of action may differ although the attenuation response indicates there may be some interaction between the two.     

Effect of retinoic acid and apo-RBP on serum retinol concentration in acute renal failure

We recently demonstrated a rapid up-regulation of serum retinol-retinol binding protein-transthyretin concentration in rats with short-term acute renal failure. We examine the effect of retinoic acid and apo-retinol binding protein (apo-RBP) on the up-regulation of serum retinol in renal failure. Injection of retinoic acid (10 micrograms) into rats with acute renal failure or sham-operated rats increased circulatory retinoic acid concentration 29-fold within 2 h but did not influence serum retinol concentration in either group. Injection of a large dose of retinoic acid (100 micrograms) decreased serum retinol concentration in rats with acute renal failure (19%) and sham-operated rats (29%). These results suggest that changes in serum retinoic acid concentration within the near-physiological range have no effect on regulation of hepatic retinol release. Injection of a large dose of retinoic acid may depress serum retinol indirectly via a retinol sparing effect in target tissues. In rats with renal failure the serum retinol concentration, elevated 44-52% above that of sham-operated controls, was also increased to 70-164% above controls by the injection of 52-63 micrograms of apo-RBP. This suggests that circulatory apo-RBP can up-regulate serum retinol. Circulatory apo-RBP may be a positive physiological feedback signal from peripheral tissues for hepatic release of retinol.     

Increased retinoic acid metabolism following 3,3',4,4',5,5'-hexabromobiphenyl injection

Young male Wistar rats received single i.p. injections of 3,3',4,4',5,5'-hexabromobiphenyl. In rats dosed with 40 mg/kg, food consumption and growth as well as liver retinol and retinyl palmitate concentrations decreased, while serum retinol and liver weight increased within 28 days following the injection. In rats receiving a 20-mg/kg dose, food consumption, growth, liver weight, and serum retinol were not affect, although liver retinol and retinyl palmitate concentrations declined to 23 and 21% of their respective control values. Vitamin A metabolism was studied in liver microsomes prepared from rats sacrificed 7 days after the 20-mg/kg injection. The rate of retinoic acid hydroxylation via the cytochrome P-450 system to 4-hydroxyretinoic acid plus the subsequent oxidation to 4-ketoretinoic acid was significantly elevated. Retinoic acid conjugation by UDP-glucuronyl transferase was also significantly increased. These changes corresponded with increased activities of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and UDP-glucuronyltransferase conjugation of p-nitrophenol. These results provide a direct link between enzyme induction due to xenobiotics and specific steps in the vitamin A metabolic pathway.     

Recent advances in the in vivo and in vitro metabolism of retinoic acid.

Retinoic acid, a natural metabolite of retinol, has previously been shown to be capable of supporting growth and maintaining proper differentiation in epithelial tissues. Recently, investigation into the in vivo and in vitro metabolism of retinoic acid in hamsters, using both tracheal organ culture and subcellular preparations derived from intestinal mucosa, liver, and testis, has revealed the production of several metabolites more polar than the parent compound. Two of the early products of this metabolic pathway have been identified as 4-hydroxy- and 4-keto-retinoic acid. The formation of these metabolites is maximal in vitamin A-deficient hamsters that have been pretreated with retinoic acid and in vitamin A-normal animals. This fact, together with the decreased biological activity of the two compounds relative to retinoic acid in a tracheal organ culture assay, suggested that oxidative attack at carbon-four of the cyclohexenyl ring may be the first step in the elimination of retinoic acid from tissues. In addition, observations both in vivo and in vitro indicate that all-trans- and 13-cis-retinoic acid at low concentrations may be sharing a common metabolic pathway that includes an isomer of 4-keto-retinoic acid.     

Pathways of absorption of retinal and retinoic acid in the rat

The chemical and anatomical pathways of absorption of dietary retinal, retinoic acid, and retinol were examined in rats containing lymph, bile, and duodenal cannulae. The experiments were designed to maintain physiological conditions to the greatest possible extent. In each rat an uninterrupted flow of bile into the duodenum was maintained by connecting the duodenal cannula to the bile duct of a second rat. Labeled vitamin A compounds were introduced into the duodenum in very small amounts (7-14 micrograms) in the form of a bile-lipid mixture resembling normal intestinal contents. Under these conditions, most (70-80%) of the radioactivity recovered after the feeding of labeled retinol or retinal was found in the lymph, predominantly in saturated retinyl esters. In contrast, 92-95% of the radioactivity recovered after the feeding of labeled retinoic acid was found in the bile, and was contained in a mixture of polar metabolites, most of them more polar than free retinoic acid. Two-thirds of the small amount of radioactivity found in lymph after retinoic acid-(14)C feeding was in the form of free retinoic acid. The results indicate that under normal conditions the major pathway of retinal absorption involves its reduction to retinol, which is then esterified and transported via the lymphatics in a manner similar to that of dietary retinol. A small proportion of retinal is apparently normally oxidized, and is then transported via the portal vein and excreted in the bile in a manner similar to that of dietary retinoic acid. The relative importance, in quantitative terms, of these two pathways of retinal metabolism can vary, depending on the status of the animal.     

Genetic evidence that retinaldehyde dehydrogenase Raldh1 (Aldh1a1) functions downstream of alcohol dehydrogenase Adh1 in metabolism of retinol to retinoic acid

Vitamin A (retinol) is a nutrient that is essential for developmental regulation but toxic in large amounts. Previous genetic studies have revealed that alcohol dehydrogenase Adh1 is required for efficient clearance of excess retinol to prevent toxicity, thus demonstrating that the mechanism involves oxidation of excess retinol to retinoic acid (RA). Whereas Adh1 plays a dominant role in the first step of the clearance pathway (oxidation of retinol to retinaldehyde), it is unknown what controls the second step (oxidation of retinaldehyde to RA). We now present genetic evidence that aldehyde dehydrogenase Aldh1a1, also known as retinaldehyde dehydrogenase Raldh1, plays a dominant role in the second step of retinol clearance in adult mice. Serum RA levels following a 50 mg/kg dose of retinol were reduced 72% in Raldh1-/- mice and 82% in Adh1-/- mice. This represented reductions in RA synthesis of 77-78% for each mutant after corrections for altered RA degradation in each. After retinol dosing, serum retinaldehyde was increased 2.5-fold in Raldh1-/- mice (indicating defective retinaldehyde clearance) and decreased 3-fold in Adh1-/- mice (indicating defective retinaldehyde synthesis). Serum retinol clearance following retinol administration was decreased 7% in Raldh1-/- mice and 69% in Adh1-/- mice. LD50 studies indicated a small increase in retinol toxicity in Raldh1-/- mice and a large increase in Adh1-/- mice. These observations demonstrate that Raldh1 functions downstream of Adh1 in the oxidative metabolism of excess retinol and that toxicity correlates primarily with accumulating retinol rather than retinaldehyde.     

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.     

Short chain dehydrogenase/reductase rdhe2 is a novel retinol dehydrogenase essential for frog embryonic development

The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Sphingosine 1-phosphate differentially regulates proliferation of C2C12 reserve cells and myoblasts

Scoliosis is a condition that involves an abnormal curvature and deformity of the spinal vertebrae. The genetic background and key gene for congenital scoliosis in humans are still poorly understood. Ishibashi rats (ISR) have congenital malformation of the lumbar vertebrae leading to kyphoscoliosis similar to that seen in humans. To understand the pathogenesis of congenital scoliosis, we have studied the abnormality of vertebral formation and the associated gene expression in ISR. Almost all ISR showed kyphosis or scoliosis of the lumbar vertebrae. In ISR with severe kyphosis, some vertebral disks were missing and some vertebral bodies were fused. Of the ISR, 27% showed hemi-lumbarization of lumbar and sacral vertebrae. Homeotic transformation of the first sacral vertebra into the seventh lumbar vertebra and the resultant loss of the fourth sacral vertebra were seen in half of the ISR. We also found unilateral fusions and deformities of primary ossification centers of the lumbar vertebral column in fetal ISR. Moreover, we observed that the expression levels of Hox10 and Hox11 paralogs in lumbo-sacral transitional areas of ISR were extremely low compared with those of normal rats. These results suggest that fusion of primary ossification centers in lumbar vertebrae in the embryonic period causes scoliosis and kyphosis and that Hox genes are involved in the occurrence of homeotic transformation in lumbo-sacral vertebrae of congenital kyphoscoliotic ISR.     

Formation of retinoic acid from retinol in the rat

1. The formation in vivo of retinoic acid from microgram quantities of intrajugularly administered [15-(14)C]retinol was demonstrated in the rat. 2. Endogenously formed retinoic acid (about 0.1mug./rat) was found in liver, and to a much smaller extent in intestine, 12hr. after retinol administration. 3. Excretion of some of the endogenously formed retinoic acid occurred in the bile of bile-duct-cannulated rats. 4. Excretion of unaltered retinoic acid in the urine of intact rats did not occur even after the intrajugular administration of preformed retinoic acid.     

Retinoic acid synthesis and functions in early embryonic development

Retinoic acid (RA) is a morphogen derived from retinol (vitamin A) that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.     

Effects of phosphorus and vitamin C deficiency,vitamin A toxicity,and lipid peroxidation on skeletal abnormalities in Atlantic halibut (Hippoglossus hippoglossus)

Dietary nutrients play an important role in skeletal tissue metabolism of fish. Deficiency and toxicity of certain nutrients have been linked to bone deformities in larval and juvenile fish. The pathogenesis of skeletal disorders in larval and juvenile fish from the same genetic stock, cultured under similar environment conditions is often difficult to distinguish when marginal deficiencies of multiple nutrients are involved. A study was conducted to characterize the skeletal deformities linked to the deficiency of phosphorus and ascorbic acid, vitamin A toxicity and lipid peroxidation in juvenile halibut. Five experimental diets containing a low level of phosphorus (0.5% dry matter basis), no vitamin C supplement, high level of vitamin A (80 000 IU kg^?1) and oxidized marine fish oil (peroxide value, 7.53 meq kg^?1) and a control diet based on cod fillet and vitamin free casein were fed to juvenile Atlantic halibut for 14 weeks in an attempt to characterize the skeletal deformities. Phosphorus, ascorbic acid, retinol, and α‐tocopherol concentrations of liver and kidney were measured at 0 and 14 weeks. Reduced vertebral ash and phosphorus content were observed in fish fed the low phosphorus diet. Skeletal abnormalities included abnormal hemal and neural spines in the hemal region and scoliosis in the cephalic and hemal regions of the vertebral column. Hepatic and kidney ascorbic acid concentrations were significantly lower in the group fed no ascorbic acid supplement. Skeletal abnormalities were scoliosis and lordosis primarily in the hemal region of the vertebral column. High levels of vitamin A in the diet caused increased hepatic retinol content and scoliosis spanning the cephalic/prehemal and anterior hemal regions of the vertebral column. Fish fed the oxidized oil diet showed increased thiobarbituric acid (TBA) value in the liver and muscle tissue with no significant decrease in hepatic vitamin E concentration. The most frequent skeletal deformity observed was scoliosis, spanning the cephalic/prehemal regions as well as the anterior hemal region of the vertebral column. The pattern and type of abnormalities observed in fish fed these experimental diets were similar to those observed in a commercial halibut hatchery.     

Metabolism of all-trans-retinol and all-trans-retinoic acid in rabbit tracheal epithelial cells in culture

As reported previously squamous cell differentiation of rabbit tracheal epithelial (RTE) cells in culture is a multi-step process. This program of differentiation is inhibited by retinoic acid and retinol; retinoic acid is about 100 times more effective than retinol. To examine the metabolism of these agents in this in vitro model system, RTE cells were grown in the presence of all-trans-[3H]retinol or all-trans-[3H]retinoic acid and their metabolites analyzed by high-pressure liquid chromatography. RTE cells converted most of the retinol to retinyl esters, predominantly retinyl palmitate. A small fraction was metabolized to polar compounds, one of which coeluted with retinoic acid. After methylation this compound eluted as 13-cis-methyl retinoate and as all-trans-methyl retinoate. Conversion to 13-cis-retinol was also observed. All-trans-retinoic acid was rapidly taken up by RTE cells and converted to more polar (peak 1) and less polar (peak 3) metabolites. A proportion of all-trans-[3H]retinoic acid was metabolized to 13-cis-[3H]retinoic acid. These metabolic reactions appeared to be constitutive and were not induced by pretreatment with retinoic acid. The peak 1 metabolites were rapidly secreted into the medium whereas the peak 3 metabolites were retained by the cells and were not detected in the medium. Alkaline hydrolysis of the metabolites in peak 3 yielded retinoic acid, indicating the formation of retinoyl derivatives. Our results establish that RTE cells can convert all-trans-retinol to 13-cis-retinol and retinoic acid. RTE can metabolize all-trans-retinoic acid to 13-cis-retinoic acid and to an unidentified ester of retinoic acid.